SMART? RACE cDNA Amplification Kit
Protocol-at-a-Glance (PT3269-2)
Please read the User Manual for the SMART RACE cDNA Amplification Kit (Cat. No. 634914) before using this Protocol-at-a-Glance. T his abbreviated protocol is provided for your convenience, but is not intended for first-time users.
First-Strand cDNA Synthesis (Section VII of the User Manual)
The two 10-μl reactions described below convert 50 ng–1 μg of total or poly A+ RNA into RACE-Ready first-strand cDNA.
1. Combine the following in separate microcentrifuge tubes:
For preparation of For preparation of
5'-RACE-Ready cDNA 3'-RACE Ready cDNA
1–3 μl RNA sample 1–3 μl RNA sample
1 μl 5'-CDS primer 1 μl 3'-CDS primer A
1 μl SMART II A oligo
2. Add sterile H2O to a final volume of 5 μl for each reaction.
3. Mix contents and spin the tube briefly in a microcentrifuge.
4. Incubate the tube at 70°C for 2 min; then cool the tube on ice for 2 min.
5. Spin the tube briefly to collect the contents at the bottom.
6. Add the following to each reaction tube (already containing 5 μl):
2 μl 5X First-Strand buffer
1 μl DTT (20 mM)
1 μl dNTP Mix (10 mM)
1 μl MMLV Reverse T ranscriptase*
10 μl T otal volume
*Please see Addendum PT3980-4 for details on the choice of RT enzyme.
7. Mix the contents and spin the tube briefly.
8. Incubate the tube at 42°C for 1.5 hr in an air incubator or a hot lid thermal cycler.
9. Dilute the first-strand reaction product with T ricine-EDTA Buffer:
? Add 20 μl if you started with <200 ng of total RNA.
? Add 100 μl if you started with >200 ng of total RNA.
? Add 250 μl if you started with poly A+ RNA.
10. Heat tubes at 72°C for 7 min.
11. Samples can be stored at –20°C for up to three months.
Positive Control PCR Experiment (Section VIII of the User Manual)
Prior to performing 5'- and 3'-RACE reactions, we strongly recommend that you per-form the positive control RACE PCR experiment in Section VIII of the User Manual.
Rapid Amplification of cDNA Ends (RACE) (Section IX of the User Manual) 1. Prepare enough PCR Master Mix for all of the PCR reactions plus one extra re-
action to ensure sufficient volume. The same Master Mix can be used for both 5'- and 3'-RACE reactions. For each 50-μl reaction, mix the following reagents:
34.5 μl PCR-Grade Water
5 μl 10X Advantage 2 PCR Buffer
1 μl dNTP Mix (10 mM)
1 μl 50X Advantage
2 Polymerase Mix
41.5 μl T otal volume
Mix well by vortexing (without introducing bubbles) and spin the tube briefly. (PR752278; published 6 June 2007)
2 Version No. PR752278
2. For 5'-RACE and 3'-RACE: prepare PCR reactions as shown in T ables III & IV. Add the components in the order shown in PCR tubes.
3. Overlay the contents of each tube with 2 drops of mineral oil and place caps firmly on each tube Note: Mineral
oil is not necessary if you are using a hot-lid thermal cycler.
* Skip this reaction if your RNA is nonhuman.
? Skip this reaction if your GSPs will not create overlapping RACE fragments.
Skip this reaction if your RNA is nonhuman.
? Skip this reaction if your GSPs will not create overlapping RACE fragments.
Version No. PR752278
3
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SMART? T echnology is covered by U.S. Patent Nos. 5,962,271 and 5,962,272. For-Profit and Not-For-Profit purchasers of SMART? Products are entitled to use the reagents for internal research. However, the following uses are expressly prohibited: (1) performing services for third parties; (2) identifying nucleic acid sequences to be included on nucleic acid arrays, blots, or in libraries or other cDNA collections which are then sold to third parties. Reproduction, modification, reformulation, or resale of the reagents provided in SMART? Products is not permitted. For information on licensing SMART? T echnology for commercial purposes, please contact a licensing representative by phone at 650.919.7320 or by e-mail at licensing@https://www.doczj.com/doc/022003568.html,.
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Clontech is a T akara Bio Company. ?2007 Clontech Laboratories, Inc. 4. Commence thermal cycling using the appropriate program for touchdown PCR. If the T m 's of your GSPs
are less than 70°C, you cannot use touchdown PCR. Consult the User Manual for an alternative PCR program. Be sure to choose the correct number of cycles (as noted) based on whether you started with poly A + or total RNA. ? 5 cycles: 94°C 30 sec 72°C 3 min ? 5 cycles: 94°C 30 sec 70°C 30 sec 72°C 3 min ? 20 cycles (Poly A + RNA): OR ? 25 cycles (Total RNA): 94°C 30 sec 68°C 30 sec 72°C 3 min